Abstract

Neutralization of the normally acidic stratum corneum (SC) has deleterious consequences for permeability barrier homeostasis and SC integrity/cohesion attributable to serine proteases (SPs) activation leading to deactivation/degradation of lipid-processing enzymes and corneodesmosomes (CD). As an elevated pH compromises SC structure and function, we asked here whether SC hyperacidification would improve the structure and function. We lowered the pH of mouse SC using two polyhydroxyl acids (PHA), lactobionic acid (LBA), or gluconolactone (GL). Applications of the PHA reduced the pH at all levels of SC of hairless mouse, with further selective acidification of SC membrane domains, as shown by fluorescence lifetime imaging. Hyperacidification improved permeability barrier homeostasis, attributable to increased activities of two key membrane-localized, ceramide-generating hydrolytic enzymes (beta-glucocerebrosidase and acidic sphingomyelinase), which correlated with accelerated extracellular maturation of SC lamellar membranes. Hyperacidification generated "supernormal" SC integrity/cohesion, attributable to an SP-dependent decreased degradation of desmoglein-1 (DSG1) and the induction of DSG3 expression in lower SC. As SC hyperacidification improves the structure and function, even of normal epidermis, these studies lay the groundwork for an assessment of the potential utility of SC acidification as a therapeutic strategy for inflammatory dermatoses, characterized by abnormalities in barrier function, cohesion, and surface pH.

Fluorescence Lifetime Imaging (FLIM) of unperturbed stratum corneum treated with LBA vs normalized LBA (nLBA), used as a control. A series of five optical sections is shown, starting at the skin surface (top) and extending to the SG (8 μm). Images are shown en face, and pH is measured by assessing the lifetime of the pH-sensitive moderator BCECF. Intensity imagines (lower two rows) are compared with lifetime images (upper two rows). Light blue represents more neutral values, whereas green and yellow represent more acidic values. Amorphous acidic collections are seen on the surface of the LBA but not on the nLBA-treated skin, likely corresponding to topically applied LBA. Owing to LBA, extracellular acidity is more visible in the LBA-treated SC, although acidification diminishes at the SG, likely representing decreased LBA diffusion at deeper SC/SG levels. Bar = 10 μm.

(a) LBA application immediately after tape stripping of normal murine skin, accelerated processing of lamellar bilayers at the stratum corneum–stratum granulosum (SC–SG) interface (shown by white arrowheads on LBA-treated sites in right panel) in comparison with nLBA-treated (left panel) 3 hours after acute barrier disruption. RuO4 post-fixation. Bar = 200 nm. (b) Accelerated maturation of lamellar bilayers to an increased in situ activity of β-GlcCer'ase and aSMase in outer SG/inner SC interface 3 hours after acute barrier disruption in comparison with nLBA treatment. The increase in β-GlcCer'ase and aSMase activities is pH-dependent and not consequent on an increase in enzyme mass, as in situ neutralization downregulates activities of both enzymes to the initial basal level.

To assess the effects of skin acidification on SC integrity/cohesion, LBA and GL were applied to hairless mice flanks. Integrity (a) markedly improved in the lower SC 3 hours after a single application of LBA and GL. Similarly, a 3-hour exposure of normal skin to either LBA or GL significantly improved SC cohesion (b), again at the lower levels of the SC. Results shown as the mean ± SEM.